Recent development of fluorescent indicator dyes for biologically important intracellular components has made it possible to follow the time-dependent distribution of these components in intact cells. For example, see U.S. Pat. Nos. 5,049,673, 4,849,362 and 4,603,208.
The emitted fluorescent radiation from a fluorophore in response to excitation at a single wavelength generally represents a broad band of wavelengths. The spectra of emissions from different fluorophores will generally overlap. This is a major problem in many practical applications involving multiple fluorophores, making quantitative measurements of the individual fluorophores difficult or impossible.
Fluorophores absorb excitation radiation at more than one wavelength. The absorption spectra of different fluorophores may thus also overlap. In summary, in the past fluorescent detection has been primarily limited to cases where the absorption spectra overlap only to a small extent or where the wavelength regions of spectral overlap of the emissions can be suppressed by optical filtering. In the latter case separation is achieved at the expense of losing valuable signal intensity.
When a fluorophore is excited by light having a sinusoidally modulated intensity the fluorescence emitted is also sinusoidally modulated. The modulation frequencies are the same but the phase of the emitted fluorescence is shifted by an amount related to the lifetime of the fluorophore's excited state. U.S. Pat. No. 4,937,457 to Mitchell discloses a frequency domain spectrofluorometer which uses a single wavelength of excitation modulated at multiple harmonically related phase-locked frequencies to simultaneously determine the spectral response and phase shift of a single fluorophore to the entire range of modulation frequencies employed. The data produced is used to determine the fluorescence lifetime of a fluorophore.
In U.S. Pat. No. 5,032,714 to Takahashi et al. a light waveform measuring device is used for measuring the lifetime of fluorescent light produced due to pulsed laser excitation. Two laser beams of different frequencies, at least one of which is pulsed, are used to produce a single-frequency pulsed beam selected from the sum frequency mixing of the beams. The output beam is pulsed at the same rate as the pulsed input beam which is used to trigger a single-photon detector or streak camera. The detector is thereby synchronized to the exciting beam.
Identification and discrimination of multiple fluorophores in a sample is disclosed in U.S. Pat. No. 5,047,321 to Loken et al. Each component must have a distinguishable characteristic peak emission wavelength at which a detector is set. Fluorophores may be excited with a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap.
In U.S. Pat. No. 4,628,026 to Gardell et al. an automated system for the sequential and alternate irradiation of a specimen by two distinguishable wavelengths of light is disclosed. The system classifies specimens based on the quotient of the fluorescent light intensities sequentially received from the specimen in response to the two excitation wavelengths.
Simultaneous recording of multiple fluorophores excited by a single wavelength using spectral filtering or separation is disclosed in U.S. Pat. No. 4,833,332 to Robertson, Jr. et al. The fluorophores which have overlapping emission spectra are distinguished by the ratio of their emissions transmitted by two spectral filters having complementary transmission spectra. The system is not capable of quantitative determinations.
The prior art devices which measure fluorescence at only one peak emission wavelength are unable to simultaneously quantitate multiple fluorophores using the total emission from each fluorophore. Those devices which rely on spectral separation to distinguish multiple fluorophores are unable to separate the total contribution of each fluorophore from the combined emission spectrum detected.
It is an object to provide an improved microfluorometer capable of simultaneously quantitating multiple fluorophores with greater efficiency.
It is another object of the present invention to provide an improved microfluorometer which simultaneously utilizes the entire emissions of multiple fluorophores or the entire emission spectra except possibly for minor parts.
It is a further object to provide an improved microfluorometer capable of separating the contribution from fluorophores having overlapping absorption spectra from the combined emission spectrum detected.